This proposal focuses on elucidating signaling pathways usurped by HIV in target cells for efficient cell-to-cell transmission. Cell-to-cell transmission involves viral assembly in infected donor cells followed by transfer to target cells through adhesive junctions called virological synapses (VS). The actin cytoskeleton is indispensable for VS mediated HIV transmission. Actin signaling through Rho-GTPases was shown to be important for efficient cell free infection. Downstream effectors of Rho-GTPases are LIMK and Arp2/3 which directly impact the actin cytoskeleton. We hypothesize that target cell interaction with HIV infected donor cells activates Rho GTPase signaling with subsequent actin cytoskeletal remodeling allowing for efficient HIV uptake. In order to interrogate this hypothesis, the following aims will be tested: 1/ Study the role of LIMK and Arp2/3 in promoting HIV cell-to-cell transmission. Our preliminary data shows that LIMK and Arp2/3 are essential for HIV cell-to-cell transfer and target cell productive infection. We intend to elucidate the upstream Rho GTPase signaling pathway activated in target cells. We also propose to dissect the specific effect of LIMK and Arp2/3 inhibition on target cell actin dynamics. 2/ Characterize the steps of viral transfer impacted by LIMK and Arp2/3 inhibition. Efficient HIV transfer from donor cells to target cells involves cessation of target cell motility, formation of adhesive interactions between infected donor and target cell populations, co-localization of host and viral factors, formation of stable cell-cell conjugates and bulk viral transfer. We hypothesize that LIMK and Arp2/3 affect transfer by impacting one or multiple of these actin dependent steps. As such, we will probe LIMK and Arp2/3 inhibition on target cell motility, adhesion, recruitment of factors essential for VS formation and stable conjugate formation. 3/ Utilize a genetic screen to determine additional actin dependent signaling pathways necessary for HIV target cell infection. We have successfully generated a T-cell line that responds to HIV cell-to-cell transfer with a reporter switch. We intend to utilize this cell line to perform a focused screen of actin dependent and membrane trafficking genes postulated to be important for target cell HIV signaling and infection.